The use of smokeless tobacco (ST) in increasing in the United States. Recent epidemiological and clinical evidence suggests that regular use of ST is associated with buccal mucosa injury, inflammation and epithelial cell dysplasia. However, the mechanisms that mediate these effects are still poorly understood. The basic tenet of this proposal is that ST induces buccal mucosa injury, inflammation and epithelial cell proliferation, in part, by altering local kinin metabolism. We hypothesized that exposure of the buccal mucosa to ST is associated with: 1) local generation and release of bradykinin (BK) that has potent inflammatory and mitogenic effects; 2) decreased activity and/or expression of the membrane-bound metalloenzymes angiotensin-converting enzymes (ACE; EC 3.4.15.1) and neutral endopeptidase (NEP; EC 23.4.24.11) that are widely distributed in the buccal mucosa and degrade BK very effectively; 3) potentiation of BK-induced buccal mucosa epithelial cell proliferation; and 4) upregulation Ki-ras oncongene expression in buccal mucosa epithelial cells leading upregulation of BK receptors in these cells. The net result would be a decrease in BK catabolism in the buccal mucosa combined with and increase in the number and/or affinity of its receptors in epithelial cells leading to potentiation of BK-induced plasm extravasation and epithelial cell proliferation. In the present proposal, we will use the hamster cheek pouch to investigate the following specific aims: 1) to determine whether ST increases vascular permeability in the hamster check pouch, and whether these effects are modulated by ACE and NEP; 2) to investigate whether ST induces BK generation and release in the hamster cheek pouch; 3) to determine whether ST decreases the activity and expression of ACE and NEP in the hamster cheek pouch; 4) to investigate whether ST interacts with BK to induce hamster cheek pouch epithelial cell DNA synthesis and proliferation in vitro, and whether these effects are modulated by ACE and NEP; and 5) to determine whether ST upregulates BK receptors in cultured hamster cheek pouch epithelial cells, and whether these effects are associated with increased expression of Ki-ras mRNA. The results of the proposed studies will provide new insights into mechanisms that may mediate the injurious effects of ST in the buccal mucosa. In the long term, they may also provide a rationale for the development of novel strategies to treat oral lesions associated with the regular use of ST in human subjects.